R-spondins-1-4 (RSPO1, RSPO2, RSPO3, and RSPO4) are secreted glycoproteins that potentiate Wnt signaling in vertebrates. RSPO actions are crucial for development and adult tissue homeostasis, and aberrant RSPO signaling is implicated in cancer. RSPOs are best characterized as growth factors for adult stem cells of the small intestine. Culture systems that permit in vitro growth of intestinal organoids from adult stem cells are dependent on exogenous RSPO1. RSPOs are thus valuable for regenerative medicine applications. Exogenous RSPO1 administration alleviated colitis symptoms in a mouse model and provided protection against chemo- and radiation therapy-induced tissue damage in mice.
RSPOs contain an N-terminal signal peptide followed by two cysteine-rich Furin-like domains, Fu1 and Fu2, a thrombospondin (TSP) domain, and a C-terminal basic region. The Fu1-Fu2 domain module is minimally sufficient to potentiate Wnt signaling. RSPOs signal through the leucine-rich repeat G protein-coupled receptors (GPCRs) LGR4-6 and the transmembrane E3 ubiquitin ligases ZNRF3 and RNF43. LGR5 marks intestinal adult stem cells. LGR4 is broadly expressed, but its co-expression with LGR5 in intestinal stem cells is crucial for their proliferation and crypt formation. RSPOs do not appear to activate classical GPCR signaling pathways; instead, RSPOs regulate Wnt receptor availability. ZNRF3/RNF43 ubiquitinate Frizzled Wnt receptors to cause their internalization and degradation. RSPOs inhibit ZNRF3/RNF43 by forming a ternary complex with LGR4/5/6 and ZNRF3/RNF43, which leads to membrane clearance of the E3 ligase and thereby more Wnt receptors at the cell surface. LGR4/5/6 have a large extracellular domain (ECD) with seventeen leucine rich-repeats (LRR) that provides the RSPO binding site, and ZNRF3/RNF43 have a small ECD for RSPO binding. Crystal structures of binary and ternary complexes and mutagenesis studies indicated that distinct regions of RSPO Fu1-Fu2 contact the two receptors.
All four RSPOs signal by a common mechanism and can promote the growth of intestinal stem cells, but significant functional differences exist. Genetic studies revealed roles for RSPO1-RSPO4 in sex determination, limb formation, placental development, and formation of toe- and fingernails, respectively. In cell-based Wnt signaling assays, RSPO2 and -3 were more active than RSPO1 and -4, and RSPO4 is considered the least active RSPO. The RSPOs bind LGRs with nM binding affinities, but conflicting results were reported for the rank order of RSPO1-RSPO4 binding to LGR4. RSPO2 and RSPO3 bound ZNRF3 with nM affinities, whereas RSPO1 and RSPO4 had affinities in the M range with RSPO4 having the weakest ZNRF3 binding.
Novel high-potency versions of RSPOs would be of value for regenerative medicine and/or therapeutic applications. It is to such novel R-spondins that the present disclosure is directed.